Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has typically been exploited by incorporating naturally occurring and genetically engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the serine proteases. Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen et al., “Homology Modelling and Protein Engineering, Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases”, Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment is frequently not optimized for the artificial environment of a cleaning composition. Specifically, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability, and substrate specificity are not necessarily optimized for utilization outside the natural environment of the protease.
Several approaches have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign and/or chemical modification of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
However, because such modified proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause an immunogenic and/or allergenic response (herein collectively described as immunogenic response) in mammals.
Furthermore, while genetic redesign and chemical modification of proteases has been prominent in the continuing search for more highly effective proteases for laundry applications, such proteases have not been commercially utilized in personal care compositions and light duty detergents. A primary reason for the absence of these proteases in products such as, for example, soaps, gels, body washes, shampoos, and light duty dish detergents is due to the problem of human sensitization leading to undesirable immunogenic responses. It would therefore be highly advantageous to provide a personal care composition or light duty detergent which provides the cleansing properties of proteases without the provocation of an immunogenic response.
Presently, immunogenic response to proteases may be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne proteases, still present the risks associated with extended tissue contact with the finished composition and worker exposure to protease-containing dust or aerosol during manufacturing.
It has also been proposed that reduction in immnunogenicity of a protease may be achieved by attaching polymers to the protease. See. e.g., U.S. Pat. No. 4,179,337, Davis et al issued Dec. 18, 1979; U.S. Pat. No. 5,856,451, Olsen et al., assigned to Novo Nordisk, issued Jan. 5, 1999; WO 99/00489, Olsen et al., assigned to Novo Nordisk, published Jan. 7, 1999; WO 98/30682, Olsen et al., assigned to Novo Nordisk, published Jul. 16, 1998; and WO 98/35026, Von Der Osten et al., published Aug. 13, 1998. However, such proposals have not suggested the importance of attaching polymers to the amino acid regions of the protease which are responsible for the immune response (ie., epitopes).
It has recently been discovered that the subtilisin protease comprises three epitope regions and that conjugation of one or more polymers, polypeptides, or other groups should be attached at one or more of these regions to effect significant reduction in immunogenicity of the protease. See, e.g., U.S. patent application Ser. No. 09/088,912, Weisgerber et al., assigned to The Procter & Gamble Co., filed Jun. 2, 1998.
The present inventors have discovered that steric protection near one or more of the epitope regions of the protease is an alternative mechanism to prevent or impede presentation of an epitope and decrease the immunogenicity of the protease. Accordingly, the present inventors herein provide modified subtilisins wherein the modification is at a region in steric proximity to one or more of the epitope regions. The present inventors have therefore discovered subtilisin proteases which evoke a decreased immunogenic response yet maintain their activity as an efficient and active protease. Accordingly, the present protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact tens compositions.